363 Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior

Cimi Ilmiawati, Kotaro Horiguchi, Ken Fujiwara and Takashi Yashiro Division of Histology and Biology, Department of Anatomy, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan (Correspondence should be addressed to T Yashiro; Email: [email protected]; C Ilmiawati; Email: [email protected])

Abstract Folliculostellate (FS) cells of the gland express RT-PCR was used to quantify MMP-9 expression in cultured a variety of regulatory molecules. Using transgenic rats that FS cells under different conditions and treatments. MMP-9 express green fluorescent protein specifically in FS cells, we expression was inhibited by pharmacological inhibitor or recently demonstrated that FS cells in vitro showed marked downregulated by siRNA and time-lapse images were changes in motility, proliferation, and that formation of cellular acquired. A 5-bromo-20-deoxyuridine assay was performed interconnections in the presence of laminin, a component of to analyze the proliferation of FS cells. Our results showed that the extracellular matrix, closely resembled those observed MMP-9 was expressed in FS cells, that this expression was in vivo. These findings suggested that FS cells express matrix upregulated by laminin, and that laminin induced MMP-9 metalloproteinase-9 (MMP-9), which assists their function on secretion by FS cells. MMP-9 inhibition and downregulation laminin. In the present study, we investigate MMP-9 did not impair FS motility; however, it did impair the capacity expression in rat anterior pituitary gland and examine its of FS cells to form interconnections and it significantly role in motility and proliferation of FS cells on laminin. inhibited proliferation of FS cells on laminin. We conclude that Immunohistochemistry, RT-PCR, immunoblotting, and gela- MMP-9 is necessary in FS cell interconnection and tin zymography were performed to assess MMP-9 expression proliferation in the presence of laminin. in the anterior pituitary gland and cultured FS cells. Real-time Journal of Endocrinology (2012) 212, 363–370

Introduction enzymes. Among the enzymes capable of ECM degradation is the matrix metalloproteinase (MMP) family. MMP-9 has The anterior pituitary gland regulates homeostasis by meticu- been extensively studied under numerous physiological and lous adjustment of hormonal secretion. Folliculostellate (FS) pathological conditions. In addition to its well-known proteo- cells are present in the anterior pituitary gland but do not secrete lytic action, MMP-9 modulates cell motility and proliferation classical hormones. Although FS cells are agranular, evidence (Moon et al.2003, Cauwe et al.2009, Sans-Fons et al.2010). suggests that they are important in coordinating anterior Despite many studies of MMP-9 at the molecular and cellular pituitary function via the homotypic cellular network through levels in various organs, MMP-9 expression and function in communication in the gland (Fauquier et al.2001, FS cells of the anterior pituitary have not been investigated. Shirasawa et al.2004). Formation of clusters and elongated Thus, the role of MMP-9 in FS cell motility and proliferation cytoplasmic processes are the structural hallmarks of FS cells under the influence of laminin needs to be clarified. (Soji & Herbert 1989). However, FS cells are known for another We investigated MMP-9 expression and localization in FS unique feature: apposition of their cytoplasmic processes and cells. In addition, we studied the effect of laminin on MMP-9 the extracellular matrix (ECM) of the basement membrane expression in primary culture and attempted to confirm the (Inoue et al.1999, Shirasawa et al.2004). role of MMP-9 in FS cell interconnection and proliferation. In a series of experiments that investigated the influence on FS cells of laminin – an ECM component of the basement membrane – we found that FS cells exhibited a motile Materials and Methods phenotype and enhanced proliferation and that they established numerous interconnections, which closely resembled their Animals cellular arrangement in vivo (Horiguchi et al.2010, 2011a,b). Transgenic S100b-green fluorescent protein (GFP) rats The high motility of FS cells on laminin suggested that, to (Itakura et al. 2007), which express GFP under the promoter migrate on the ECM, they must express matrix-degrading control of the S100b protein gene (a marker of FS cells), were

Journal of Endocrinology (2012) 212, 363–370 DOI: 10.1530/JOE-11-0433 0022–0795/12/0212–363 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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donated by Prof. K Inoue of Saitama University and bred in RNase-free DNase I (1 U/tube; Promega Corp.) and heated our laboratory. Eight- to ten-week-old male rats weighing to inactivate DNase I. cDNA was synthesized using a 250–300 g were given ad libitum access to food and water and Superscript III RT kit with oligo-(dT)20 primer (Invitrogen). housed under a 12 h light:12 h darkness cycle. Rats were The PCR mix consisted of the RT reaction product, PCR sacrificed by exsanguination from the right atrium under buffer containing dNTPs, KOD Dash DNA Polymerase C deep Nembutal anesthesia and then perfused with Ca2 - and (2.5U/ml; Toyobo, Osaka, Japan), and each oligonucleotide C Mg2 -free Hanks’ solution for primary culture or with primer. The primer pairs used and putative product lengths 4% paraformaldehyde (PFA) in 0.05 M phosphate buffer were as follows: MMP-9 (GenBank accession no. (pH 7.4) for immunohistochemistry. All animals were treated NM_031055), forward: 50-AGG GTC GGT TCT GAC in accordance with the Jichi Medical University Guidelines CTT TT-30, reverse: 50-TGA GGG ATC ATCc TCG GCT for Animal Experimentation. AC-30 (522 bp); S100b (BC_087026), forward: 50-ATA GCA CCT CCG TTG GAC AG-30, reverse: 50-CAT CTC AGT 0 Cell culture GGC CCT TCA TT-3 (527 bp); and glyceraldehyde-3- phosphate dehydrogenase (GAPDH; M_17701), forward: Anterior pituitary cells of S100b-GFP male rats were 50-CCA TCA CCA TCT TCC AGG AG-30,reverse:50-TTC dispersed as described in a previous report (Horiguchi et al. AGC TCT GGG ATG ACC TT-30 (457 bp). 2008). The dispersed cells were then sorted by a MoFlo XDP Quantitative real-time PCR (ABI PRISM 7900HT; (Beckman Coulter, Inc., Fullerton, CA, USA) into GFP- Applied Biosystems, Carlsbad, CA, USA) was performed by C K positive (GFP ) and GFP-negative (GFP ) cell fractions. using gene-specific primers and SYBR Premix Ex Taq C GFP cells were plated onto eight-well glass chamber slides (Takara, Tokyo, Japan) containing SYBR Green I. To amplify (1 cm2/well; Nalge Nunc International, Rochester, NY, 2 cDNA fragments, the following primers were used: MMP-9, USA), with or without a coating of 10 mg/cm laminin 0 0 ! 5 2 forward: 5 -CCT GAA AAC CTC CAA CCT CA-3 , (Millipore, Bedford, MA, USA), at a density of 1 10 cells/cm reverse: 50-GGA CTG CTT CTC TCC CAT CA-30 (100 bp); in 400 ml of Medium 199 with Earle’s salts (Invitrogen) 0 cyclin D1 (NM_171992.4), forward: 5 -TGC AAA TGG supplemented with 10% fetal bovine serum (Sigma–Aldrich 0 0 AAC TGC TTC TG-3 , reverse: 5 -GCG GAT GAT Corp.), 0.5 U/ml penicillin, and 0.5 mg/ml streptomycin CTG CTT GTT CT-30 (125 bp). As reference, we also (Invitrogen). Cells were then incubated at 37 8Cinan quantified GAPDH, forward: 50-AAG GGC TCA TGA atmosphere of 5% CO and 95% air. For MMP-9 inhibition, 2 CCA CAG TC-30, reverse: 50-GGA TGC AGG GAT GAT cells were treated with 100 mM synthetic MMP-9/MMP-13 GTT CT-30 (116 bp). Relative quantification was conducted Inhibitor I (444252; Merck), and control groups were treated using the standard curve method and was performed with an equal volume of dimethyl sulfoxide 9 (DMSO); in triplicate. (Wako, Osaka, Japan) in the medium from 0 h. For MMP-9 knockdown, a siRNA sequence against rat MMP-9 (SI102004247; Qiagen), with a nontargeting siRNA Immunoblot analysis (1027283; Qiagen) as control, was transfected into cells by Anterior pituitary gland, GFPC and GFPK cell fractions using an INTERFERin transfection reagent (PolyPlus C Transfections, Inc., New York, NY, USA) 24 h after seeding. separated by cell sorter and primary cultured GFP cells Cells were time-lapse recorded using a digital camera were lysed in RIPA buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% w/v SDS, 1% v/v Triton X-100; (ORCA-ER; Hamamatsu Photonics, Shizuoka, Japan) and . MetaMorph Software (Molecular Devices Corp., Downingtown, pH 7 5), and total protein was estimated by Bradford assay (Sigma). Twenty micrograms of protein from each sample PA,USA)from2to72hafterseedinginaCO2 gas culture chamber (Sankei Corp., Tokyo, Japan) with a thermostat were applied to 10% SDS–PAGE. Proteins were then (Kokensha Engineering Corp., Tokyo, Japan) on a fluor- transferred electrophoretically onto Immobilon-P transfer escence-inverted microscope (IX71; Olympus Corp., Tokyo, membranes (Millipore). Membranes were blocked with Japan). Each observation was performed in triplicate. 5% skim milk in TBST (50 mM Tris, 100 mM NaCl, 0.1% v/v Tween 20; pH 7.4), probed overnight at room temperature with rabbit monoclonal anti-MMP-9 antibody RT-PCR and real-time RT-PCR diluted 1:15 000 (ab76003; Abcam, Inc.) or mouse anti- For RT-PCR, total RNA fractions were prepared with b-actin antibody (0.1 mg/ml; BioVision, Mountain View, TRIzol reagent (Invitrogen) from anterior pituitary gland, CA, USA), diluted in Can Get Signal solution (Toyobo) primary culture of anterior pituitary cells, and the GFPC and followed by TBST washes, and incubated for 1 h with GFPK cell fractions of S100b-GFP male rats. Expression of HRP-labeled secondary antibodies (EnvisionCSystem– MMP-9 and cyclin D1 mRNA in primary cultured FS cells HRP, anti-rabbit; Dako, Glostrup, Denmark). Immuno- was measured by real-time RT-PCR. Total RNA fractions reactive bands were visualized by ECL Plus Western Blotting were prepared with the RNeasy Mini kit (Qiagen) from Detection Reagents (GE Healthcare, Mississauga, ON, cultured FS cells, and all RNA samples were incubated with Canada). Each blot was performed in triplicate.

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Gelatin zymography laser microscope with a 60-fold objective lens. Observations were done in triplicate for each experimental group. Gelatin zymography to detect MMP-9 activity was per- formed according to the method described in a previous report (Zhang & Gottschall 1997). In brief, protein samples Statistical analysis from anterior pituitary gland and GFPC and GFPK cell Results are presented as meanGS.E.M. Student’s t-test was fractions were prepared using nonreducing sample buffer used to compare differences between groups, which were containing 0.5 M Tris–HCl (pH 6.8), 10% SDS, and glycerol. considered to be statistically significant at a P value of !0.05. GFPC cells were cultured as described above for 48 h. Then, medium was replaced with serum-free medium supple- mented with 0.1% BSA (Roche Diagnostics), and cell culture was continued for 24 h. Conditioned medium was collected AB from each well of three experimental replicates and centrifuged at 15 000 g for 3 min at 4 8C to remove cellular AL IL PL AL IL PL debris. Samples were electrophoretically run in copolymer- ized 10% SDS–acrylamide (Bio-Rad Laboratories, Inc.) and 0.1% gelatin (Difco Laboratories, Detroit, MI, USA) gel. Gel was incubated in regeneration buffer containing 2.5% Triton C DE X-100 for 2 h at room temperature followed by incubation in reaction buffer containing 10 mM CaCl2 for 24 h at 37 8C. To visualize gelatinolytic bands, the gel was stained with Coomassie Brilliant Blue (ATTO Corp., Tokyo, Japan).

Immunohistochemistry F G After perfusion with 4% PFA in 0.05 M PB (pH 7.4), the GFP+ GFP+ 10 Tissue GFP– GFP+ uncoated laminin * pituitary glands were carefully dissected and fixed overnight in MMP–9 8 S100b 6 GAPDH the same fixative solution at 4 8C. Next, tissues were 4 . . (fold change) 2 immersed in 0 05 M PB (pH 7 2) containing 30% sucrose level MMP-9 mRNA 0 for 2 days at 4 8C, embedded in Tissue-Tek OCT compound Uncoated Laminin H I (Sakura Finetechnical, Tokyo, Japan), and snap-frozen. GFP+ GFP+ GFP+ GFP+ Tissue GFP– GFP+ uncoated laminin GFP– GFP+ uncoated laminin Frontal sections (8 mm) were incubated in PBS containing 92 kDa MMP–9 82 kDa MMP–9 82 kDa 2% normal goat serum for 20 min at 30 8C to block β-Actin 42 kDa nonspecific antigen binding, then incubated with anti-rat Figure 1 MMP-9 expression in FS cells. (A) Pituitary cryosection MMP-9 rabbit monoclonal antibody diluted 1:25 in PBS for from S100b-GFP rat stained with hematoxylin–eosin showing the two nights at 4 8C, followed by incubation with Alexa anterior lobe (AL), intermediate lobe (IL), and posterior lobe (PL). Fluor 568-conjugated goat anti-rabbit IgG diluted 1:200 Scale bar, 100 mm. (B) Pituitary cryosection from S100b-GFP rat showing GFPC cells in anterior lobe (AL), intermediate lobe (IL), (Invitrogen) for 30 min at 30 8C. For immunostaining of and posterior lobe (PL). In anterior lobe, S100b expression (green) is cultured GFPC cells, cells were fixed with 4% PFA in limited to FS cells. Scale bar, 100 mm. (C) S100b-GFP rat anterior 0.025 M PB (pH 7.4) for 20 min at room temperature, pituitary showing clusters of GFPC FS cells. (D) MMP-9 immersed in PBS containing 2% normal goat serum for immunoreactivity (red) in anterior pituitary section. (E) Overlay image of (C) and (D), MMP-9 immunoreactivity (red) is observed in 20 min at 30 8C, incubated with anti-rat MMP-9 rabbit most GFPC cells (green). Scale bars for C-E, 10 mm. (F) Expression monoclonal antibody diluted 1:100 overnight at room of MMP-9 gene in anterior pituitary. Total RNA fractions extracted temperature, then probed with Alexa Fluor 568-conjugated from anterior pituitary tissue (tissue), GFPK and GFPC cell C goat anti-rabbit IgG. Absence of an observable nonspecific fractions (by cell sorter), and primary culture of GFP cells on uncoated (GFPC uncoated) and laminin-coated (GFPC laminin) reaction was confirmed using normal rabbit serum instead of surfaces were analyzed by RT-PCR of MMP-9, S100b, and GAPDH. primary antibody. (G) Expression of MMP-9 mRNA as determined by real-time RT-PCR after 72 h incubation on uncoated (uncoated) and laminin-coated (laminin) surfaces, normalized to internal control (GAPDH; Proliferation assay meanGS.E.M., nZ3, *P!0.05). (H) Immunoblotting showing MMP-9 expression in anterior pituitary tissue (tissue), GFPK and To observe FS cell proliferation, the nucleotide analog GFPC cell fractions, and primary cultured GFPC cells on uncoated 5-bromo-20-deoxyuridine (BrdU; Sigma) was incorporated (GFPC uncoated) and laminin-coated (GFPC laminin) surfaces. into primary culture and detected in fixed cells according to (I) Gelatin zymography showing MMP-9 activity in anterior pituitary cell fractions (GFPK, GFPC) and conditioned medium of FS cells previously described procedures (Horiguchi et al. 2010). (GFPC) cultured on uncoated (GFPC uncoated) and laminin- Thirty random fields were imaged per well using a confocal coated (GFPC laminin) surfaces. www.endocrinology-journals.org Journal of Endocrinology (2012) 212, 363–370

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Results cells from the anterior pituitary of S100b-GFP rats. These cells were then cultured on uncoated and laminin-coated MMP-9 expression in anterior pituitary gland chamber slides for 72 h. The shape of FS cells differed when grown on uncoated and laminin-coated surfaces (Fig. 2A–D; Localization of MMP-9 in anterior pituitary gland was Horiguchi et al. 2010). On the uncoated surface, FS cells investigated immunohistochemically. Figure 1Ashows acquired a round shape and aggregated (Fig. 2A and B). hematoxylin–eosin staining of S100b-GFP rat pituitary Under the influence of laminin, FS cells appeared to flatten gland. Transgenic S100b-GFP rats expressed GFP specifically and form interconnections (Fig. 2C and D). Immunocyto- in FS cells of the anterior pituitary (Fig. 1B). Frozen frontal chemistry to detect MMP-9 distribution demonstrated that sections of S100b-GFP rat pituitary tissue were probed with FS cells showed punctate MMP-9 immunoreactivity on the anti-rat MMP-9 rabbit monoclonal antibody (Fig. 1C and D). uncoated surface (Fig. 2B). However, under the influence of MMP-9 immunoreactivity was observed throughout the laminin, MMP-9 had a filament-like distribution on FS cells anterior pituitary, specifically in clusters of GFPC cells and in (Fig. 2D). their long cytoplasmic processes (Fig. 1E). Most FS cells showed MMP-9 immunoreactivity on their cell bodies and cytoplasmic extensions. Next, expression of MMP-9 gene in Effect of MMP-9 inhibitor on FS cells in primary culture FS cells was examined by RT-PCR. MMP-9 was detected in To determine the role of MMP-9 on FS cells in the presence anterior pituitary cells both in vivo and in vitro (Fig. 1F). of laminin, we observed the behavior of living FS cells in Analysis of GFPC and GFPK cell fractions showed identical primary culture with MMP-9 inhibitor (Fig. 3A and B). Cells results, with the exception of the S100b, which was not were treated with 100 mM MMP-9 synthetic inhibitor or an detected in the GFPK cell fraction (Fig. 1F). Real-time equal volume of vehicle (DMSO; control group) from 0 to RT-PCRwas performed to quantify the relative expression of 72 h. In the control group, the behavior of FS cells was similar MMP-9 in FS cells cultured on uncoated and laminin-coated to that reported previously (Horiguchi et al. 2010, 2011a), surfaces (Fig. 1G). Expression of MMP-9 was markedly i.e. by 72 h of culture, they attached to laminin by becoming higher in GFPC cells cultured on laminin than in those flatter, showed remarkable locomotion, extended their cultured on an uncoated surface (P!0.05). cytoplasm toward other cells in their vicinity, and formed Expression of MMP-9 was confirmed by immunoblot interconnections with other FS cells (Fig. 3A; Supplementary analysis of anterior pituitary cell lysate in vivo and in vitro.We data Movie 1, see section on supplementary data given at the detected an immunoreactive band at 82 kDa, which end of this article). In the group treated with MMP-9 corresponds to the size of activated MMP-9 in anterior pituitary tissue (Fig. 1H). Blotting of the GFPC cell fraction protein showed a strong immunoreactive band, correspond- A B ing to the 82 kDa protein, and a weaker band of 92 kDa that corresponds to the molecular weight of latent MMP-9 (Fig. 1H). No immunoreactive band appeared in the sample of the GFPK cell fraction (Fig. 1H). GFPC cells of primary culture on uncoated surface showed very weak immuno- reactive bands. However, two bands of MMP-9 appeared for GFPC cells on laminin-coated primary culture (Fig. 1H). MMP-9, also known as 92 kDa gelatinase B, can digest gelatin, i.e. denatured collagen. Gelatin zymography was performed to detect this specific property of MMP-9. C D Protein samples were collected with nonreducing sample buffer from GFPK and GFPC cell fractions. The only clear gelatinolytic band for the GFPC cell fraction corresponded to 82 kDa (Fig. 1I). We then investigated whether FS cells secreted MMP-9 into the media. Serum-free media of FS cells cultured in the absence or presence of laminin were collected. The clear gelatinolytic band corresponding to 82 kDa was stronger for medium from FS cells conditioned in the presence of laminin (Fig. 1I). Figure 2 Immunocytochemistry of MMP-9 in primary cultured FS cells. (A and C) Phase contrast images. (B) Confocal image of FS MMP-9 localization on FS cells in primary culture cells cultured on uncoated surface. (D) Confocal image of FS cells cultured on laminin-coated surface. Note that MMP-9 immuno- To observe MMP-9 localization on isolated FS cells in reactivity (red) is higher in FS cells (green) cultured on laminin- primary culture, a cell sorter was used to separate GFPC FS coated surface. Scale bar, 10 mm.

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2 h 24 h 48 h 72 h A

G B 1

0·5 FS cells

** Ratio of BrdU-positive 0 C D E F Control MMP-9 inhibitor

Figure 3 Time-lapse images of FS cells isolated from S100b-GFP rat anterior pituitary cells in primary culture on laminin-coated surface, with or without MMP-9 inhibitor. Cells are time-lapse recorded at 15 min intervals from 2 to 72 h after plating. (A and B) GFP images superimposed on phase contrast images, using an inverted fluorescence microscope, on laminin-coated surface without (A) and with (B) MMP-9 inhibitor. 2, 24, 48, and 72 h: elapsed time from plating of cells. Higher magnification views are shown in the inset images. Scale bar, 100 mm. (C–F) BrdU incorporation into FS cells. (C and D) Control group treated with DMSO. BrdU immunoreactivity (red) is observed in FS cells. (E and F) Representative FS cells under the influence of MMP-9 inhibitor show very little proliferative activity. Scale bar, 10 mm. (G) Ratio of BrdU-positive FS cells cultured on laminin-coated surfaces in the absence (control) or presence of MMP-9 inhibitor (MMP-9 inhibitor; meanGS.E.M., nZ3, **P!0.01). inhibitor, FS cells did not fully flatten but did extend their interconnections was also diminished, resulting in small, cytoplasmic processes. In addition, although the motility of scattered clusters by 72 h of culture (Fig. 4B, 72 h). FS these cells was comparable to that of the control group, they cells transfected for an identical period with nontargeting only established sparse cellular interconnections by 72 h of siRNA reconstructed interconnections (Fig. 4A, 72 h; culture (Fig. 3B; Supplementary data Movie 2, see section on Supplementary data Movie 3, see section on supplementary supplementary data given at the end of this article). To data given at the end of this article). Quantitative real-time investigate whether MMP-9 affects the proliferative capacity RT-PCR analysis of MMP-9 expression showed that siRNA of FS cells, we performed BrdU incorporation in FS cells transfection decreased MMP-9 gene expression as compared cultured without or with MMP-9 inhibitor. Figure 3C and D with control (P!0.01; Fig. 4C). We also examined the effect shows BrdU incorporation in FS cells cultured on the of MMP-9 silencing on FS cell proliferation and found that laminin-coated surface in the absence of MMP-9 inhibitor. In MMP-9 downregulation decreased the ratio of BrdU-positive ! . the presence of MMP-9 inhibitor (Fig. 3E and F), a marked FS cells as compared with control (P 0 01; Fig. 4D). decrease in BrdU-positive cells was observed. As compared However, MMP-9 silencing did not significantly change the with the control group, the ratio of BrdU-positive cells was expression of cyclin D1 (Fig. 4E). lower on the laminin-coated surface with MMP-9 inhibitor (P!0.01; Fig. 3G). Discussion Knockdown of MMP-9 gene by siRNA This study shows that MMP-9 is specifically expressed in The use of siRNA to silence the expression of MMP-9 FS cells of normal adult rat pituitary, that its expression is resulted in a marked change in FS cell shape. By 24 h upregulated by laminin, and that it is involved in laminin- of culture, plasma membrane projections were already mediated morphological and proliferative changes in FS established in most FS cells (Fig. 4A and B, 24 h). Transfection cells in vitro. with MMP-9 siRNA from 24 to 72 h after plating led to To clarify the mechanisms underlying the marked motility retraction of these projections; i.e. FS cells adopted a round and proliferation of FS cells on laminin, we investigated shape and detached from laminin (Fig. 4B, 48, 72 h; MMP-9 expression in rat anterior pituitary and isolated Supplementary data Movie 4, see section on supplementary FS cells, based on the techniques of our earlier study data given at the end of this article). The number of cellular (Horiguchi et al. 2010). We found that FS cells express www.endocrinology-journals.org Journal of Endocrinology (2012) 212, 363–370

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2 h 24 h 48 h 72 h A

B

E C D 1·5 1·5 1·5

1·0 1·0 1·0

** FS cells * 0·5 0·5 0·5 (fold change) (fold change) MMP-9 mRNA level MMP-9 mRNA Ratio of BrdU-positive Cyclin D1 mRNA level Cyclin D1 mRNA 0 0 0 Control MMP-9 Control MMP-9 Control MMP-9 siRNA siRNA siRNA Figure 4 Time-lapse images of FS cells (in primary culture on laminin-coated surface) transfected with nontargeting siRNA or MMP-9 siRNA. (A and B) GFP images superimposed on phase contrast images on laminin-coated surface with nontargeting siRNA (A) and MMP-9 siRNA (B). 2, 24, 48, and 72 h: elapsed time from plating of cells. Higher magnification views are shown in the inset images. Scale bar, 100 mm. (C) Quantitative real-time RT-PCR shows that MMP-9 gene expression level is significantly lower in FS cells transfected with MMP-9 siRNA (MMP-9 siRNA) than in those transfected with nontargeting siRNA (control); results are normalized to internal control (GAPDH; meanGS.E.M., nZ3, **P!0.01). (D) Ratio of BrdU-positive FS cells, cultured on laminin-coated surfaces, transfected with nontargeting siRNA (control) or MMP-9 siRNA (MMP-9 siRNA; *P!0.05). (E) Quantitative real-time RT-PCR shows that cyclin D1 expression is not affected by silencing MMP-9 expression; results are normalized to internal control (GAPDH; meanGS.E.M., nZ3).

MMP-9 mRNA and protein (Fig. 1D and F). Other studies (Horiguchi et al. 2010). Since laminin induces integrin b1 have used immunohistochemistry (Knappe et al. 2003) and signaling in FS cells (Horiguchi et al. 2011b), we hypothesize zymography (Paez Pereda et al. 2000) to investigate MMP-9 that the increase in MMP-9 expression in the presence expression in normal and tumorous human anterior of laminin occurs via a similar pathway. Furthermore, the pituitary glands. However, those earlier studies did not increases in MMP-9 mRNA expression and protein (in cell identify MMP-9-expressing cells in the gland. lysate and secreted into medium) in the presence of laminin We also observed that FS cells differentially expressed parallel the phenotypic change in FS cell shape from round to MMP-9 in the absence and presence of laminin and that this stellate (Figs 1D–G and 2). Punctate MMP-9 immuno- expression was increased by laminin (Fig. 1D–G). In several reactivity was observed in round FS cells on the uncoated cell lines, laminin-derived peptide (Freitas et al. 2007) and surface. In contrast, filament-like signals were seen in clusters whole laminin (Maity et al. 2011) induced MMP-9 secretion of stellate-shaped FS cells on laminin (Fig. 2). MMP-9 was and activity upon interaction with integrin by utilizing the spatially distributed along microfibers (Schumacher et al. ERK pathway. The integrins comprise an a-subunit and 2005), and elevated MMP-9 expression was found to be b-subunit, which form a heterodimer. In mammals, 18 types associated with the invasive phenotype of cells (Peters et al. of a-subunits and eight types of b-subunits are known; their 1999). Sbai et al. (2010) showed that MMP-9 has a vesicular various combinations give rise to 24 integrin heterodimers, distribution and that the location of vesicles is associated with which differ in ligand specificity (Hynes 2002). With respect the cytoskeleton. Previously, we found that motile FS cells on to these ligand specificities, we recently reported that FS cells laminin formed F-actin in their cytoplasmic processes during bind laminin through integrin-a3b1 and/or integrin-a6b1 cellular interconnection (Horiguchi et al. 2010). It is thus

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Downloaded from Bioscientifica.com at 09/23/2021 10:07:42PM via free access MMP-9 expression in FS cells . C ILMIAWATI and others 369 possible that the differential spatial distribution of MMP-9 network (Soji et al. 1997). It is tempting to speculate that under the influence of laminin is related to variation in the MMP-9 contributes to the expansion of this FS cell network pattern of cytoskeletal arrangement in FS cells. in vivo. Future study is required to identify the mechanisms To determine the role of MMP-9 in FS cells, we used involved in the network arrangement of FS cells. MMP-9 inhibitor to observe the behavior of living FS cells in primary culture. Inhibition of MMP-9 abrogated FS cell interconnection on laminin (Fig. 3; Supplementary data Supplementary data Movie 2, see section on supplementary data given at the end This is linked to the online version of the paper at http://dx.doi.org/10.1530/ of this article). Because MMP-13 is a secondary target for the JOE-11-0433. inhibitor used in this study, we next ruled out the effect of MMP-13 inhibition on our observations by silencing MMP-9 expression. The results were similar to those Declaration of interest observed with MMP-9 inhibitor, i.e. FS cells failed to reconstruct dense interconnections, due to loss of attachment The authors hereby declare that there is no conflict of interest that could be to laminin (Fig. 4; Supplementary data Movie 4, see section perceived as prejudicing the impartiality of the research reported in the manuscript entitled ‘Matrix metalloproteinase-9 expression in folliculostellate on supplementary data given at the end of this article). cells of rat anterior pituitary gland’. The interconnections between FS cells are believed to serve as a network for conveying cellular messages through gap junctions in the anterior pituitary gland (Soji & Herbert Funding 1989, Fauquier et al. 2001, Shirasawa et al. 2004). Previously, we discovered that FS cells formed networks in the presence This work was partially supported by a Grant-in-Aid for Scientific Research b (C) (22590192), a Grant-in-Aid for Young Scientists (B) (22790190) from the of laminin, a process mediated through integrin 1 signaling Ministry of Education, Culture, Sports, Science and Technologyof Japan, and (Horiguchi et al. 2011a). Veeravalli et al. (2010) showed that by promotional funds from the Keirin Race of the Japan Keirin Association. MMP-9 silencing downregulates the a-subunit and b-subunit of integrin in vitro. Knocking down MMP-9 expression might inhibit integrin signaling in FS cells and therefore influence Acknowledgements adhesion of FS cells to laminin. Our present results confirm that FS cells do at least partly depend on MMP-9 expression We thank Prof. Kinji Inoue (Saitama University, Japan) for supplying the transgenic rats. We are grateful to Miss Megumi Yatabe for her excellent to interconnect and assemble their characteristic network on technical assistance and to Prof. Yutaka Hanazono and Mr Yutaka Furukawa laminin. for their support in fluorescence-activated cell sorting. We also thank David MMP-9 promotes cell proliferation in several cell types Kipler, ELS of Supernatant Communications for revising the language of the (Dwivedi et al. 2009, Sans-Fons et al. 2010, Ingraham et al. manuscript. 2011). The present study showed that MMP-9 inhibition suppressed proliferative activity of FS cells on laminin, which indicates that MMP-9 is necessary in FS cell division (Figs 3G and 4D). Proliferation of FS cells on laminin involves References integrin b1, which is associated with caveolin-3, and uses MAPK signal transduction, which upregulates the cyclin D1 Cauwe B, Martens E, Proost P & Opdenakker G 2009 Multidimensional degradomics identifies systemic autoantigens and intracellular matrix expression that drives the cell cycle (Horiguchi et al. 2011b). proteins as novel gelatinase B/MMP-9 substrates. Integrative Biology 1 Despite the marked decrease in BrdU incorporation after 404–426. (doi:10.1039/b904701h) MMP-9 silencing, cyclin D1 expression was unchanged in Dwivedi A, Slater SC & George SJ 2009 MMP-9, and -12 cause N-cadherin this study (Fig. 4). This unexpected finding suggests that shedding and thereby beta-catenin signalling and vascular smooth muscle MMP-9 does not affect the capacity of cells to progress from cell proliferation. Cardiovascular Research 81 178–186. (doi:10.1093/cvr/ cvn278) G1 to S phase via the caveolin-3-mediated signaling pathway, Fauquier T, Guerineau NC, McKinney RA, Bauer K & Mollard P 2001 but rather that MMP-9 is necessary for FS cells to synthesize Folliculostellate cell network: a route for long-distance communication in DNA. We are continuing to investigate the role of MMP-9 in the anterior pituitary. PNAS 98 8891–8896. (doi:10.1073/pnas. FS cell proliferation. 151339598) In conclusion, the present study provides evidence that Freitas VM, Vilas-Boas VF, Pimenta DC, Loureiro V, Juliano MA, Carvalho MR, Pinheiro JJ, Camargo AC, Moriscot AS, Hoffman MP et al. laminin promotes MMP-9 expression in FS cells of rat 2007 SIKVAV, a laminin alpha1-derived peptide, interacts with integrins anterior pituitary gland and that MMP-9 is required for the and increases protease activity of a human salivary gland adenoid cystic interconnection and proliferation of FS cells. In the presence carcinoma cell line through the ERK 1/2 signaling pathway. American of laminin, increased MMP-9 expression appears to promote Journal of Pathology 171 124–138. (doi:10.2353/ajpath.2007.051264) the characteristic features of FS cells in anterior pituitary Horiguchi K, Fujiwara K, Kouki T, Kikuchi M & Yashiro T 2008 Immunohistochemistry of connexin 43 throughout anterior pituitary gland gland. During the early postnatal period, a small number of FS in a transgenic rat with green fluorescent protein-expressing folliculo- cells appear sparsely in the anterior pituitary at day 10; during stellate cells. Anatomical Science International 83 256–260. (doi:10.1111/j. maturation, they gradually form a more interconnected cell 1447-073X.2008.00239.x) www.endocrinology-journals.org Journal of Endocrinology (2012) 212, 363–370

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